Research in plant molecular biology has advanced dramatically in recent years and is necessary for the analysis of various physiological phenomena. Dwarfism caused by artificial modification of grass type, especially the control of elongation growth, prevents plants from lodging due to overgrowth caused by over fertilization. This prevention of lodging was demonstrated in Mexican wheat during the “Green Revolution” and in miracle rice (IR-8) developed by the International Rice Research Center. Furthermore, in the case of cultivation at high density, such as rice cultivation, yields are expected to increase as a result of the increase in the amount of sun light each plant receives due to the formation of upright leaves. Moreover, these modifications are very important breeding targets because they may result in yield increases and also increase the efficiency of plant growth maintenance. However, current breeding methods cannot artificially modify plant morphology.
Dwarfism is an abnormal growth caused by mutation in genes involved in controlling normal elongation growth. Plant elongation growth is the result of accumulation of cell division and cell elongation. Cell division and cell elongation are controlled by complex effects caused by various factors, such as, exogenous environmental factors including temperature and light and endogenous environmental factors including plant hormones. Therefore, it is predicted that many genes, such as those related to plant hormone biosynthesis and hormone receptors directly and those related to the control of the expression of these genes, are involved in the dwarfism (Sakamoto et al. (2000) Kagaku to Seibutsu, 38: 131–139).
Almost all modern cultivars of japonica rice develop 15–16 phytomers, consisting of leaves, axillary buds, and short or elongated internodes, during the vegetative stage. After the shoot meristem shifts from the vegetative to the reproductive phase, the reproductive meristem develops about 10 phytomers consisting of undeveloped leaf, an elongated internode, and an axillary which develops into the primary rachis branch. The phytomers formed in the vegetative stage can be classified into three types in terms of the morphology of the internode (Suetsugu, Isao. (1968) Japan. J. Crop Sci. 37, 489–498). The first type is developed in the juvenile phase and form undifferentiated nodes and internodes. After the shoot apical meristem (SAM) shifts from the juvenile to the adult phase, the nodal plate of the second type differentiates and the central part of the internode thereof decays to produce an air space. The third type contains long elongated internodes as a result of growth from the intercalary meristem.
Phytomers of type 1 are produced first during vegetative development, followed by type 2 and then type 3 phytomers. Under normal growth conditions, the number of phytomers of each type in many japonica cultivars is 4–5, 6–7, and 4–5. The transition from type 1 to type 2 is strictly regulated. After the serial development of 4–5 type 1 phytomers, depending on the cultivar, the SAM develops type 2 phytomers. However, the transition from type 2 to type 3 does not depend on the number of development of type 2 phytomers. The SAM develops 15–16 phytomers and shifts from the vegetative to the reproductive stage, the type 3 phytomers then start to develop, and the uppermost four or five internodes thereof start to elongate. If the timing of the transition is changed by unusual growth conditions, the number of the type 2 phytomers always affected thereby, but the number of the type 3 phytomer with elongated internode is unchanged (Suetsugu, isao. (1968) Japan. J. Crop Sci: 37, 489–498). This indicates that the transition of the SAM from the vegetative to the reproductive phase in rice induces internode elongation, as well as in Arabidopsis. 
However, there is an important difference between rice and Arabidopsis. The elongated internodes in rice are derived from the vegetative SAM while those in Arabidopsis come from the reproductive SAM. In rice, the uppermost four or five internodes develop from the vegetative SAM and initially are indistinguishable from the lower type 2 internodes. When the SAM shifts to the reproductive phase, differentiation into type 3 internodes occurs due to the development of intercalary meristems in the internodes. This synchronicity between the phase change of the SAM and the development of the intercalary meristem leads to the possibility that these processes might be linked by a signal coming from the SAM to the uppermost four or five phytomers when its phase change occurs.
A large number of dwarf mutants of rice have been collected and characterized because of their agronomic importance. These dwarf mutants are categorized into six groups based on the elongation pattern of the upper four to five internodes (FIG. 1; redrawn from Takeda, K. (1974) Bull. Fac. Agr. Hirosaki Univ. 22, 19–30. In rice, each internode is numbered from top to bottom such that the uppermost internode just below the panicle is first). The present inventors can see that in the dn-type mutants the length of each internode is almost uniformly reduced, resulting in an elongation pattern similar to that of the wild type plant. In contrast, the dm-type mutants show specific reduction of the second internode. Similar shortening of a specific internode is also observed in the sh- and d6-type mutants, in which only the uppermost first internode or internodes below the uppermost are shortened, respectively. As these mutants with specifically shortened internodes, such as the dm-, d6-, and sh-types, might be defective in the perception of signals coming from the SAM, they should be especially useful for the study of the mechanism of internode elongation and its relationship to changes in the SAM.
Brassinosteroids (BRs) are plant growth-promoting natural products that are required for plant growth and development. There are only a few reports on the physiological effects of brassinosteroids in the growth and development of rice and other plants of the Gramineae family. Physiological researches indicate that exogenous brassinosteroids alone, or in combination with auxin, enhance bending of the lamina joint in rice. The lamina joint has been used for a sensitive bioassay of brassinosteroids (Maeda, E. (1965) Physiol. Plant. 18, 813–827; Wada, K. et al. (1981) Plant and Cell Physiol. 22, 323–325; Takeno, K. and Pharis, R. P. (1982) Plant Cell Physiol. 23, 1275–1281), because of high sensitivity thereof to brassinosteroids. In etiolated wheat seedlings treatment with brassinolide or its derivative, castasterone, stimulates unrolling of the leaf blades (Wada, K. et al. (1985) Agric. Biol. Chem. 49, 2249–2251). Treatment with low or high concentrations of brassinosteroids promotes or inhibits the growth of roots in rice, respectively (Radi, S. H. and Maeda, E. (1988) J. Crop Sci. 57, 191–198). Brassinosteroids also promote the germination of rice seeds (Yamaguchi, T. et al. (1987) Stimulation of germination in aged rice seeds by pre-treatment with brassinolide. In: Proceeding of the fourteenth annual plant growth regulator society of America Meeting Honolulu. (Cooke A R), pp. 26–27).
Although these results indicate only effects due to exogenous brassinosteroids, not due to endogenous brassinosteroids, they do suggest that endogenous brassinosteroids have an important role in growth and developmental processes in plants of the Gramineae family.
On the other hand, there is some apparent disagreement in the literature as to whether brassinosteroids induce cell elongation in plants of the Gramineae family. That is, brassinolide treatment does not induce elongation of the leaf sheath of rice (Yokota, T. and Takahashi, N. (1986) Chemistry, physiology and agricultural application of brassinolide and related steroids. In: Plant growth substances 1985. (Bopp M, Springer-Verlag, Berlin/Heidelberg/New York) pp.129–138), but it does induce elongation of the coleoptile and mesocotyl in maize (He, R. —Y. et al. (1991) Effects of brassinolide on growth and chilling resistance of maize seedlings. In: Brassinosteroids-Chemistry, Bioactivity and Applications ACS symposium series 474. (Cutler H G C, Yokota T, Adam G, American Chemical Society, Washington D.C.), pp. 220–230).
As shown by brassinosteroids synthesis mutants or brassinosteroids insensitive mutants that show severe dwarfism with abnormal development of organs, the function of brassinolide is known in dicotyledonous plants.
However, little is known about the function of endogenous brassinosteroids in monocotyledonous plants, such as rice or other plants of the Gramineae family.
3. Disclosure of the Invention
The object of the present invention is to provide novel genes involved in brassinosteroid sensitivity from plants, preferably from monocotyledonous plants. Another object of the present invention is to modify plant brassinosteroid sensitivity by controlling the expression of the gene. The modification in plant brassinosteroid sensitivity causes a change in plant morphology. The preferable embodiment of the present invention provides plants with erect leaves which become dwarfed due to the suppression of internode elongation caused by decreased brassinosteroid sensitivity.
By treatment with mutagenesis agent, the present inventors isolated a novel rice dwarf mutant strain d61 (d61-1 and d61-2) which showed lower brassinosteroid sensitivity and had shorter internodes than wild type plants.
Linkage analysis indicated that the d61 locus was highly linked to a gene region that was homologous to Arabidopsis BRI1. The present inventors isolated the gene (OsBRI1), which was homologous to Arabidopsis BRI1 gene, by screening of a rice genomic DNA library. Nucleotide sequence analysis of the OsBRI1 gene from d61-1 and d61-2 mutants indicated that there were single nucleotide substitutions causing amino acid substitutions at different sites in each d61 allele.
Moreover, in order to confirm that the OsBRI1 gene corresponds to the d61 locus, the OsBRI1 gene was introduced into d61 mutants. As a result, the OsBRI1 gene complimented the d61 phenotype and caused the mutant strain to have a wild-type phenotype. Therefore, it was indicated that d61 mutants are caused by loss of function of the OsBRI1 gene. Phenotypic analysis of plants revealed that the OsBRI1 gene functions in various growth and development processes of rice including internode elongation caused by formation of intercalary meristem and induction of internode cell longitudinal elongation, inclination of the lamina joint, and skotomorphogenesis in the dark.
Moreover, in the case where transgenic rice plants with OsBRI1 antisense nucleotide were produced, most transgenic plants produced erect leaves during seedling growth. All of the transgenic plants showed dwarf phenotype of various levels. Plants transformed with OSBRI1 having the dominant negative phenotype showed the same result.
The present invention had been made in view of such findings, and relates to a novel gene involved in plant brassinosteroid sensitivity, the protein encoded by the gene, and production and use of the same. Moreover, the present invention relates to the production of modified plant by controlling expression of the gene.
More specifically, this invention provides:    (1) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 2;    (2) the DNA of (1), wherein the DNA is a cDNA or a genomic DNA;    (3) the DNA of (1), wherein the DNA comprises a coding region of the nucleotide sequence of SEQ ID NO: 1 or 3;    (4) a DNA encoding a protein which has 55% or more homology to the amino acid sequence of SEQ ID NO: 2 and which is functionally equivalent to a protein comprising the amino acid sequence of SEQ ID NO: 2, the DNA being selected from the group consisting of            (a) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids are substituted, deleted, added, and/or inserted; and        (b) a DNA hybridizing under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3;            (5) the DNA of (4), wherein the DNA encodes a protein having a function selected from the group consisting of a function of increasing brassinosteroid sensitivity in a plant, a function of inducing elongation of internode cells of a stem of a plant, a function of positioning microtubules perpendicular to the direction of elongation in an internode of a stem of a plant, a function of suppressing elongation of an internode of a neck of a plant, and a function of increasing inclination of a lamina of a plant;    (6) the DNA of (4) or (5), wherein the DNA is derived from a monocotyledonous plant;    (7) the DNA of (6), wherein the DNA is derived from a plant of the Gramineae family;    (8) a DNA encoding an antisense RNA complementary to a transcript of the DNA of any one of (1) to (7);    (9) a DNA encoding an RNA having ribozyme activity which specifically cleaves a transcript of the DNA of any one of (1) to (7);    (10) a DNA which encodes an RNA repressing expression of the DNA of any one of (1) to (7) due to co-suppression when expressed in a plant cell and which has 90% or more homology to the DNA of any one of (1) to (7);    (11) a DNA which encodes a protein having a dominant negative phenotype to that of a protein encoded by the DNA of any one of (1) to (7);    (12) a vector which comprises the DNA of any one of (1) to (7);    (13) a transformed cell which comprises the DNA of any one of (1) to (7) or the vector of (12);    (14) a protein encoded by the DNA of any one of (1) to (7);    (15) a method for producing the protein of (14) the method comprising the steps of culturing the transformed cell of (13) and recovering an expressed protein from the transformed cell or a culture supernatant thereof;    (16) a vector comprising the DNA of any one of (8) to (11);    (17) a transformed plant cell comprising the DNA of any one of (1) to (11) or the vector of (12) or (16);    (18) a transformed plant comprising the transformed plant cell of (17);    (19) a transformed plant which is a progeny or a clone of the transformed plant of (18);    (20) a breeding material of the transformed plant of (18) or (19); and    (21) an antibody which binds to the protein of (14).
The present invention provides a DNA encoding the OsBRI1 protein derived from rice. The nucleotide sequence of OsBRI1 cDNA is shown in SEQ ID NO: 1, the amino acid sequence of the protein encoded by the DNA is shown in SEQ ID NO: 2, and the nucleotide sequence of the genomic DNA of OsBRI1 is shown in SEQ ID NO: 3 (the genomic DNA of SEQ ID NO: 3 consists of one exon with no intron).
The gene of the present invention causes a rice dwarf mutant (d61) which has short internodes and reduced brassinosteroid sensitivity compared to the wild type. Therefore, it is possible to modify plant morphology by controlling the expression of the OsBRI1 gene.
The preferable modification in plant morphology in the present invention includes dwarfism of plants by suppressing expression of the DNA of the present invention. Dwarfism of plants has great value in agriculture and horticulture. For example, reduction of height of plants can reduce the tendency of plants to lodge and can thereby increase seed weights. Moreover, it is possible to increase the number of plant individuals which can be planted per unit area by reducing height of plants and by making plant shape per plant more compact. These plant modifications have great value specifically in the production of crops such as rice, corn, wheat, and such. It is also possible to produce ornamental plants with new aesthetic value by dwarfism of height or culm length of plants. It is also possible to produce miniature vegetables or fruits with new commercial value, such as “bite-size”, by dwarfism of them. Other than for industrial plants, dwarfism is important for experimental plants because, for example, dwarf plants are not only more easily handled but they also help utilize experimental space more effectively by decreasing cultivation space.
It is possible to consider that brassinosteroid sensitivity can be increased in brassinosteroid low sensitive plants by expressing the DNA of the present invention in the plants. Thereby, the yield of whole plants may be increased by growing taller plants. Thus, this will be especially useful for increasing yield for whole feed crops.
DNA encoding the OsBRI1 protein of the present invention includes genomic DNA, cDNA, and chemically synthesized DNA. A genomic DNA and cDNA can be prepared according to conventional methods known to those skilled in the art. More specifically, a genomic DNA can be prepared, for example, as follows: (1) extract genomic DNA from plant cells or tissues; (2) construct a genomic library (utilizing a vector, such as plasmid, phage, cosmid, BAC, PAC, and such); (3) spread the library; and (4) conduct colony hybridization or plaque hybridization using a probe prepared based on the DNA encoding a protein of the present invention (e.g., SEQ ID NO: 1 or 3). Alternatively, a genomic DNA can be prepared by PCR, using primers specific to a DNA encoding the protein of the present invention (e.g. SEQ ID NO: 1 or 3). On the other hand, cDNA can be prepared, for example, as follows: (1) synthesize cDNAs based on mRNAs extracted from plant cells or tissues; (2) prepare a cDNA library by inserting the synthesized cDNA into vectors, such as λZAP; (3) spread the cDNA library; and (4) conduct colony hybridization or plaque hybridization as described above. Alternatively, cDNA can be also prepared by PCR.
The present invention includes DNAs encoding proteins functionally equivalent to the OsBRI1 protein of SEQ ID NO: 2. Herein, the term “functionally equivalent to the OsBRI1 protein” means that the object protein has equal functions to those of the OsBRI1 protein of SEQ ID NO: 2, such as, for example, a function of increasing brassinosteroid sensitivity in a plant, a function of inducing elongation of an internode of a stem of a plant, a function of positioning microtubules perpendicular to the direction of elongation in internode cells of a stem of a plant, a function of suppressing elongation of an internode of a neck of a plant, and/or a function of increasing inclination of a lamina of a plant. Such DNA is derived preferably from monocotyledonous plants, more preferably from plants of the Gramineae family, and most preferably from rice.
Examples of such DNAs include those encoding mutants, derivatives, alleles, variants, and homologues comprising the amino acid sequence of SEQ ID NO: 2 wherein one or more amino acids are substituted, deleted, added, and/or inserted.
Examples of methods for preparing a DNA encoding a protein comprising altered amino acids well known to those skilled in the art include the site-directed mutagenesis (Kramer, W. and Fritz, H. -J. (1987) “Oligonucleotide-directed construction of mutagenesis via gapped duplex DNA.” Methods in Enzymology, 154: 350–367). The amino acid sequence of a protein may also be mutated in nature due to the mutation of a nucleotide sequence. A DNA encoding proteins having the amino acid sequence of a natural OsBRI1 protein (SEQ ID NO: 2) wherein one or more amino acids are substituted, deleted, and/or added are also included in the DNA of the present invention, so long as they encode a protein functionally equivalent to the natural OsBRI1 protein. Additionally, nucleotide sequence mutants that do not give rise to amino acid sequence changes in the protein (degeneracy mutants) are also included in the DNA of the present invention. The number of nucleotide mutations of the DNA of interest corresponds to, at amino acid level, typically 100 residues or less, preferably 50 residues or less, more preferably 20 residues or less, and still more preferably 10 residues or less (for example, 5 residues or less, or 3 residues or less).
Whether a certain DNA actually encodes a protein which has a function of increasing inclination of a lamina of a plant can be evaluated, for example, by performing a “lamina joint test” for plants in which the expression of the DNA has been suppressed and by comparing the results with those for wild-type plants (See Example 4). The result of the test may also be an index for evaluating brassinosteroid sensitivity in a plant. In order to evaluate whether the DNA encodes a protein which has a function of inducing elongation of an internode of a stem of a plant, a function of positioning microtubules perpendicular to the direction of elongation in internode cells of a stem of a plant, or a function of suppressing elongation of an internode of a neck of a plant, the morphology of the internode cell of the plant in which expression of the DNA has been suppressed can be observed to be compared with that of wild type (See Examples 2 and 3).
A DNA encoding a protein functionally equivalent to the OsBRI1 protein described in SEQ ID NO: 2 can be produced, for example, by methods well known to those skilled in the art including: methods using hybridization techniques (Southern, E. M. (1975) Journal of Molecular Biology, 98, 503); and polymerase chain reaction (PCR) techniques (Saiki, R. K. et al. (1985) Science, 230, 1350–1354; Saiki, R. K. et al. (1988) Science, 239, 487–491). That is, it is routine for a person skilled in the art to isolate a DNA with high homology to the OsBRI1 gene from rice and other plants using the OsBRI1 gene (SEQ ID NO: 1 or 3) or parts thereof as a probe, and oligonucleotides hybridizing specifically to the gene as a primer. Such DNA encoding proteins functionally equivalent to the OsBRI1 protein, obtainable by hybridization techniques or PCR techniques, are included in the DNA of this invention.
Hybridization reactions to isolate such DNAs are preferably conducted under stringent conditions. Stringent hybridization conditions of the present invention include conditions such as: 6 M urea, 0.4% SDS, and 0.5×SSC; and those which yield a similar stringency with the conditions. DNAs with higher homology are expected to be isolated efficiently when hybridization is performed under conditions with higher stringency, for example, 6 M urea, 0.4% SDS, and 0.1×SSC. Those DNAs isolated under such conditions are expected to encode a protein having a high amino acid level homology with OsBRI1 protein (SEQ ID NO: 2). Herein, “high homology” means an identity of at least 55% or more, more preferably 70% or more, and most preferably 90% or more (e.g., 95% or more), between full-length of amino acids.
The degree of homology of one amino acid sequence or nucleotide sequence to another can be determined by following the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 90: 5873–5877, 1993). Programs such as BLASTN and BLASTX were developed based on this algorithm (Altschul et al. J. Mol. Biol. 215: 403–410, 1990). To analyze a nucleotide sequences according to BLASTN based on BLAST, the parameters are set, for example, as score=100 and word length=12. On the other hand, parameters used for the analysis of amino acid sequences by the BLASTX based on BLAST include, for example, score=50 and word length=3. Default parameters of each program are used when using BLAST and Gapped BLAST program. Specific techniques for such analysis are known in the art.
The DNA of the present invention can be used, for example, to prepare recombinant proteins, produce transformed plants with phenotypes altered by controlling expression thereof as described above, and so on.
A recombinant protein is usually prepared by inserting a DNA encoding a protein of the present invention into an appropriate expression vector, introducing said vector into an appropriate cell, culturing the transformed cells, and purifying expressed proteins.
A recombinant protein can be expressed as a fusion protein with other proteins so as to be easily purified, for example, as a fusion protein with maltose binding protein in Escherichia coli (New England Biolabs, USA, vector pMAL series), as a fusion protein with glutathione-S-transferase (GST) (Amersham Pharmacia Biotech, vector pGEX series), or tagged with histidine (Novagen, pET series). The host cell is not limited so long as the cell is suitable for expressing the recombinant protein. It is possible to utilize yeasts or various animal, plant, or insect cells besides the above described E. coli. A vector can be introduced into a host cell by a variety of methods known to one skilled in the art. For example, a transformation method using calcium ions (Mandel, M. and Higa, A. (1970) Journal of Molecular Biology, 53, 158–162; Hanahan, D. (1983) Journal of Molecular Biology, 166, 557–580) can be used to introduce a vector into E. coli. A recombinant protein expressed in host cells can be purified and recovered from the host cells or the culture supernatant thereof by known methods. When a recombinant protein is expressed as a fusion protein with maltose binding protein or other partners, the recombinant protein can be easily purified by affinity chromatography.
The resulting protein can be used to prepare an antibody that binds to the protein. For example, a polyclonal antibody can be prepared by immunizing immune animals, such as rabbits, with a purified protein of the present invention or its portion, collecting blood after a certain period, and removing clots. A monoclonal antibody can be prepared by fusing myeloma cells with the antibody-forming cells of animals immunized with the above protein or its portion, isolating a monoclonal cell expressing a desired antibody (hybridoma), and recovering the antibody from the cell. The obtained antibody can be utilized to purify or detect a protein of the present invention. Accordingly, the present invention includes antibodies that bind to proteins of the invention.
In order to produce a transformed plant in which DNAs of the present invention are expressed, a DNA encoding a protein of the present invention is inserted into an appropriate vector; the vector is then introduced into a plant cell; and finally, the resulting transformed plant cell is regenerated.
On the other hand, a transformed plant with suppressed expression of DNAs of the present invention can be created using DNA that represses the expression of a DNA encoding a protein of the present invention: wherein the DNA is inserted into an appropriate vector, the vector is introduced into a plant cell, and then, the resulting transformed plant cell is regenerated. The phrase “suppression of expression of DNA encoding a protein of the present invention” includes suppression of gene transcription as well as suppression of translation into protein. It also includes not only the complete inability of expression of DNA but also reduction of expression.
The expression of a specific endogenous gene in plants can be repressed by methods utilizing antisense technology, the methods which are commonly used in the art. Ecker et al. were the first to demonstrate the antisense effect of an antisense RNA introduced by electroporation in plant cells by using the transient gene expression method (J. R. Ecker and R. W. Davis (1986) Proc. Natl. Acad. Sci. USA 83: 5372). Thereafter, the target gene expression was reportedly reduced in tobacco and petunias by expressing antisense RNAs (A. R. van der Krol et al. (1988) Nature 333: 866). The antisense technique has now been established as a means to repress target gene expression in plants.
Multiple factors are required for antisense nucleic acid to repress the target gene expression. These include, inhibition of transcription initiation by triple strand formation; suppression of transcription by hybrid formation at the site where the RNA polymerase has formed a local open loop structure; transcription inhibition by hybrid formation with the RNA being synthesized; suppression of splicing by hybrid formation at the junction between an intron and an exon; suppression of splicing by hybrid formation at the site of spliceosome formation; suppression of mRNA translocation from the nucleus to the cytoplasm by hybrid formation with mRNA; suppression of splicing by hybrid formation at the capping site or at the poly A addition site; suppression of translation initiation by hybrid formation at the binding site for the translation initiation factors; suppression of translation by hybrid formation at the site for ribosome binding near the initiation codon; inhibition of peptide chain elongation by hybrid formation in the translated region or at the polysome binding sites of mRNA; and suppression of gene expression by hybrid formation at the sites of interaction between nucleic acids and proteins. These factors repress the target gene expression by inhibiting the process of transcription, splicing, or translation (Hirashima and Inoue, “Shin Seikagaku Jikken Koza (New Biochemistry Experimentation Lectures) 2, Kakusan (Nucleic Acids) IV, Idenshi No Fukusei To Hatsugen (Replication and Expression of Genes)”, Nihon Seikagakukai Hen (The Japanese Biochemical Society), Tokyo Kagaku Dozin, pp. 319–347, (1993)).
An antisense sequence of the present invention can repress the target gene expression by any of the above mechanisms. In one embodiment, if an antisense sequence is designed to be complementary to the untranslated region near the 5′ end of the gene's mRNA, it will effectively inhibit translation of a gene. It is also possible to use sequences complementary to the coding regions or to the untranslated region on the 3′ side. Thus, the antisense DNA used in the present invention includes DNA having antisense sequences against both the untranslated regions and the translated regions of the gene. The antisense DNA to be used is connected downstream from an appropriate promoter, and, preferably, a sequence containing the transcription termination signal is connected on the 3′ side. The DNA thus prepared can be transfected into the desired plant by known methods. The sequence of the antisense DNA is preferably a sequence complementary to the endogenous gene of the plant to be transformed or a part thereof, but it need not be perfectly complementary so long as it can effectively inhibit the gene expression. The transcribed RNA is preferably at least 90%, and most preferably at least 95% complementary to the transcribed products of the target gene. Sequence complementarity may be determined using the above-described search.
In order to effectively inhibit the expression of the target gene by means of an antisense sequence, the antisense DNA should be at least 15 nucleotides long, preferably at least 100 nucleotides long, and more preferably at least 500 nucleotides long. The antisense DNA to be used is generally shorter than 5 kb, and preferably shorter than 2.5 kb.
DNA encoding ribozymes can also be used to repress the expression of endogenous genes. A ribozyme is an RNA molecule that has catalytic activity. There are many ribozymes having various activities. Research on ribozymes as RNA cleaving enzymes has enabled the design of a ribozyme that site-specifically cleaves RNA. While some ribozymes of the group I intron type or the mRNA contained in RNaseP consist of 400 nucleotides or more, others belonging to the hammerhead type or the hairpin type have an activity domain of about 40 nucleotides (Makoto Koizumi and Eiko Ohtsuka, (1990) Tanpakushitsu Kakusan Kohso (Nucleic acid, Protein, and Enzyme), 35: 2191).
The self-cleavage domain of a hammerhead type ribozyme cleaves at the 3′ side of C15 of the sequence G13U14C15. Formation of a nucleotide pair between U14 and A at the ninth position is considered important for the ribozyme activity. Furthermore, it has been shown that the cleavage also occurs when the nucleotide at the 15th position is A or U instead of C (M.. Koizumi et al., (1988) FEBS Lett. 228: 225). If the substrate binding site of the ribozyme is designed to be complementary to the RNA sequences adjacent to the target site, one can create a restriction-enzyme-like RNA cleaving ribozyme which recognizes the sequence UC, UU, or UA within the target RNA (M. Koizumi et al., (1988) FEBS Lett. 239: 285; Makoto Koizumi and Eiko Ohtsuka, (1990) Tanpakushitsu Kakusan Kohso (Protein, Nucleic acid, and Enzyme), 35: 2191; M. Koizumi et al., (1989) Nucleic Acids Res. 17: 7059). For example, in the coding region of the OsBRI1 gene (SEQ ID NO: 1 or 3), there is a plurality of sites that can be used as the ribozyme target.
The hairpin type ribozyme is also useful in the present invention. A hairpin type ribozyme can be found, for example, in the minus strand of the satellite RNA of tobacco ringspot virus (J. M. Buzayan, Nature 323: 349 (1986)). This ribozyme has also been shown to target-specifically cleave RNA (Y. Kikuchi and N. Sasaki, (1992) Nucleic Acids Res. 19: 6751; Yo Kikuchi, (1992) Kagaku To Seibutsu (Chemistry and Biology) 30: 112).
The ribozyme designed to cleave the target is fused with a promoter, such as the cauliflower mosaic virus 35S promoter, and with a transcription termination sequence, so that it will be transcribed in plant cells. However, if extra sequences have been added to the 5′ end or the 3′ end of the transcribed RNA, the ribozyme activity can be lost. In this case, one can place an additional trimming ribozyme, which functions in cis to perform the trimming on the 5′ or the 3′ side of the ribozyme portion, in order to precisely cut the ribozyme portion from the transcribed RNA containing the ribozyme (K. Taira et al. (1990) Protein Eng. 3: 733; A. M. Dzaianott and J. J. Bujarski (1989) Proc. Natl. Acad. Sci. USA 86: 4823; C. A. Grosshands and R. T. Cech (1991) Nucleic Acids Res. 19: 3875; K. Taira et al. (1991) Nucleic Acid Res. 19: 5125). Multiple sites within the target gene can be cleaved by arranging these structural units in tandem to achieve greater effects (N. Yuyama et al. (1992) Biochem. Biophys. Res. Commun. 186: 1271). By using such ribozymes, it is possible to specifically cleave the transcripts of the target gene in the present invention, thereby repressing the expression of said gene.
Endogenous gene expression can also be repressed by co-suppression through the transformation by DNA having a sequence identical or similar to the target gene sequence. “Co-suppression” refers to the phenomenon in which, when a gene having a sequence identical or similar to the target endogenous gene sequence is introduced into plants by transformation, expression of both the introduced exogenous gene and the target endogenous gene becomes repressed. Although the detailed mechanism of co-suppression is unknown, it is frequently observed in plants (Curr. Biol. (1996) 7: R793 (1997), Curr. Biol. 6: 810). For example, if one wishes to obtain a plant body in which the OsBRI1 gene is co-repressed, the plant in question can be transformed with a vector DNA designed so as to express the OsBRI1 gene or DNA having a similar sequence to select a plant having the OsBRI1 mutant character, e.g., a plant with suppressed internode elongation, among the resultant plants. The gene to be used for co-suppression does not need to be completely identical to the target gene, but it should have at least 70% or more sequence identity, preferably 80% or more sequence identity, and more preferably 90% or more (e.g., 95% or more) sequence identity. Sequence identity may be determined by above-described search.
In addition, endogenous gene expression in the present invention can also be repressed by transforming the plant with a gene having the dominant negative phenotype of the target gene. Herein, “a DNA encoding the protein having the dominant negative phenotype” refers to a DNA encoding a protein which, when the DNA is expressed, can eliminate or reduce the activity of the protein encoded by the endogenous gene of the present invention inherent to the plant. Preferably, it is a DNA encoding the peptide (e.g., peptide which contains from 739 to 1035 residues of amino acids of SEQ ID NO: 2 or peptides of another protein equivalent to the peptide) which lacks the N-terminal region but contains the kinase region of the protein of the present invention. Whether the DNA of interest has the function to eliminate or enhance activity of the endogenous gene of the present invention can be determined, as mentioned above, by whether the DNA of interest eliminates or reduces a function of increasing brassinosteroid sensitivity in a plant, a function of inducing elongation of an internode of a stem of a plant, a function of positioning microtubules perpendicular to the direction of elongation in internode cells of a stem of a plant, a function of suppressing elongation of an internode of a neck of a plant, and/or a function of increasing inclination of a lamina of a plant.
Vectors used for the transformation of plant cells are not limited as long as the vector can express inserted genes in plant cells. For example, vectors comprising promoters for constitutive gene expression in plant cells (e.g., califlower mosaic virus 35S promoter); and promoters inducible by exogenous stimuli can be used. The term “plant cell” used herein includes various forms of plant cells, such as cultured cell suspensions, protoplasts, leaf sections, and callus.
A vector can be introduced into plant cells by known methods, such as the polyethylene glycol method, electroporation, Agrobacterium mediated transfer, and particle bombardment. Plants can be regenerated from transformed plant cells by known methods depending on the type of the plant cell (Toki et al., (1995) Plant Physiol. 100:1503–1507). For example, transformation and regeneration methods for rice plants include: (1) introducing genes into protoplasts using polyethylene glycol, and regenerating the plant body (suitable for indica rice cultivars) (Datta, S. K. (1995) in “Gene Transfer To Plants”, Potrykus I and Spangenberg Eds., pp66–74); (2) introducing genes into protoplasts using electric pulse, and regenerating the plant body (suitable for japonica rice cultivars) (Toki et al (1992) Plant Physiol. 100, 1503–1507); (3) introducing genes directly into cells by the particle bombardment, and regenerating the plant body (Christou et al. (1991) Bio/Technology, 9: 957–962); (4) introducing genes using Agrobacterium, and regenerating the plant body; and so on. These methods are already established in the art and are widely used in the technical field of the present invention. Such methods can be suitably used for the present invention.
Once a transformed plant, wherein the DNA of the present invention is introduced into the genome, is obtained, it is possible to gain descendants from that plant body by sexual or vegetative propagation. Alternatively, plants can be mass-produced from breeding materials (for example, seeds, fruits, ears, tubers, tubercles, tubs, callus, protoplast, etc.) obtained from the plant, as well as descendants or clones thereof. Plant cells transformed with the DNA of the present invention, plant bodies including these cells, descendants and clones of the plant, as well as breeding materials obtained from the plant, its descendant and clones, are all included in the present invention.
The plant of the present invention is preferably a monocotyledonous plant, more preferably a plant of the Gramineae family, and most preferably a rice. The phenotype of the plant of the present invention is different from the wild type phenotype. The phenotypes changed in the plants developed by the present invention include brassinosteroid sensitivity of a plant, plant growth such as internode cell elongation of the plant stem and internode elongation of the ear, inclination of leaves, and the positioning of microtubules perpendicular to the direction of internode cell elongation in the plant stem.